As used herein, certain citations to references are indicated as numerals or alphanumerical symbols in parentheticals, and are further described in the “References Cited” listing contained herein.
Breast cancer is the most common malignancy in women, as about a million new cases of this cancer are diagnosed worldwide each year, and 375,000 women die from it (B1). Although early detection through screening mammography has increased the proportion of in situ and early stage breast cancers that have excellent prognosis, mortality rates from recurrent and late stage breast cancers have not declined significantly (B2). Consequently, conventional therapies including surgery, radiation and chemotherapy need to be supplemented with novel therapies that translate into significant improvement in the clinical outcome for most breast cancer patients.
Similarly, prostate cancer is the most commonly diagnosed malignancy in men, with about 220,000 new cases of prostate cancer diagnosed each year in the U.S. alone. Of these, approximately two-thirds are treated by surgery or radiation therapy, and 40% of the treated men will eventually relapse, as characterized by rising levels of prostate specific antigen (B3, B4). Relapsed advanced and metastatic prostate cancer remains the primary cause of death from this cancer, since current systemic hormonal and chemotherapy approaches are only marginally successful (B5). Consequently there is an urgent need for novel therapies to treat advanced prostate cancer.
Further, despite intense therapeutic efforts, ovarian cancer remains the fifth leading cause of death among women in the United States (B6). Serous adenocarcinomas account for approximately 80% of all ovarian cancers and are considered surface epithelial tumors (B7). Early stage epithelial ovarian cancer has an excellent prognosis with 5 year survival rates above 90%. However, about 80% of patients with ovarian cancer initially present with metastatic disease, resulting in 5 year survival rates of only 10-20%. Following primary therapy, as many as 70% of women with advanced ovarian cancer will experience a recurrence of their disease. Novel therapies to eradicate the residual or recurrent malignancy are critically needed to increase the unacceptable low survival rates (B8).
Prostate derived Ets factor (PDEF) belongs to the Ets family of transcription factors that play an important role in normal as well as neoplastic development (B9-B13). However, despite such promise, knowledge in the art about the characteristics of PDEF expression in human cancer remains limited.
PDEF was originally described as a novel prostate specific transcription factor based on its prostate restricted expression in normal human tissues and its ability to induce the expression of prostate specific antigen (PSA) in tumor cell lines (A1). Shortly thereafter, it was shown that (i) PDEF mRNA was frequently over expressed in primary breast tumors from patients, and (ii) among normal tissues, it showed expression in the trachea/bronchus tissues in addition to the normal prostate tissue (A2). In contrast, other normal tissues including brain, heart, kidney, liver, lung, skeletal muscle, spleen, testis, and uterus were negative for PDEF expression (A2). More recently, a screen of a much larger collection of breast tumors and normal human tissues has confirmed the Inventor's findings and showed that roughly 75% of newly diagnosed breast tumors show varying level of over expression of PDEF (A3). These observations suggested a role for PDEF in breast and prostate cancer progression.
A major deficiency of the above studies was that all the work was performed at the PDEF mRNA level, and in the absence of any evidence showing correspondence between PDEF mRNA and PDEF protein expression, a putative role for PDEF in tumor progression remained speculative, at best. Moreover, two groups prepared antibodies against the full-length PDEF protein and reported that PDEF protein was strongly expressed in normal breast (A4) and prostate (A5) tissues, but only weakly in the tumor tissues. The latter results were diametrically opposite to those reported for PDEF mRNA (A1-3). One explanation for such discrepant reports of PDEF mRNA and protein expression was that the antibodies to full-length PDEF proteins were not specific for PDEF, and may be cross-reacting with other members of the Ets transcription factor family to which PDEF belongs. Specifically, there are two structural motifs within the sequence of PDEF, called “Pointed domain” and DNA binding “Ets” domain, and these show high level of sequence homology with similar structural motifs present in other members of the Ets factor family (FIG. 1), and these appeared likely as targets for cross-reaction with anti-PDEF antibodies.
One approach to investigating PDEF has involved the attempt to develop antibodies with the bind specifically to PDEF with high affinity. However, prior to the present invention, no such high affinity anti-PDEF antibody had been described that could be used for clinical diagnostics. For example, previously, anti-PDEF antibodies have been generated against the full-length PDEF protein (4). While these antibodies reacted with PDEF in the Western blot assay, the specificity of their reactivity in the immunohistochemistry assay for screening PDEF expression in patient's samples has remained suspect; since those antibodies reacted strongly with normal breast tissues and weakly with breast tumors tissue, a finding contradictory to previously reported PDEF mRNA expression data (3, 22, A3). A likely explanation for such uncharacteristic reactivity of antibodies against full-length PDEF is the homology between PDEF and other Ets factors in the Pointed domain and DNA binding Ets domain (see FIGS. 1B and 1C respectively). Such high degree of homology within these domains suggested the potential for cross-reaction of the antibodies with other members of the Ets transcription factor family. In fact, the Inventor's lab also previously prepared antibody against the full-length PDEF protein and found that it reacted uncharacteristically strongly with normal breast tissue.
Antibodies to fragments of PDEF have also been described, but none of these antibodies was shown to have high affinity to PDEF. For example, antibodies to amino acid residues 1-104 of PDEF were described by Ghadersohi et al. (A7, A8). However, like previously reported antibodies to full-length PDEF, the antibodies described by Ghadersohi et al. also reacted strongly with normal breast and ovarian tissues, and weakly or not at all with tumor tissues, showing a lack of specificity for PDEF in the immunohistochemical assay.
It is shown that about 80% of primary breast tumors, 70% of primary prostate tumors, and 30 to 35% of primary ovarian tumors show over-expression of PDEF protein in tumor tissues in comparison to normal tissues (1, 2). Further, (i) transfection of PDEF into a low-malignancy breast epithelial cell line induced its accelerated tumor growth in vivo in immunodeficient mice; and (ii) meta analysis of the GEO and Oncomine databases that contain the global gene expression data from breast tumors and the linked clinical outcome data for patients showed that high PDEF expression correlates with poor overall survival for patients with estrogen receptor-positive (ER+) breast tumors. In prostate cancer as well, high PDEF expression was found to correlate with early biochemical PSA failure, a surrogate marker of cancer recurrence and disease progression.
Despite considerable progress in early detection of breast and prostate cancers, mortality from advanced and metastatic disease has not declined significantly over the past several decades. Further, even with the knowledge of PDEF's role in various types of cancers, prognostic, diagnostic, and treatment methods that directed to detection of PDEF or blocking of PDEF have not been forthcoming.
The present invention is directed to the deficiencies in the art.